The invention concerns new cationic and polycationic amphiphiles which are capable of forming aggregates with macromolecules, in particular with DNA or RNA, and it concerns their delivery into prokaryotic or eukaryotic cells.
Amphiphiles (e.g. surfactants, detergents) have for some time played a general role in daily life. In contrast only recently has greater attention been paid to cationic amphiphiles through the pioneer work of Felgner since they can be used to deliver DNA and RNA into cells and transfect them in this manner (P. L. Felgner et al., Proc. Natl. Acad. Sci., USA 84, 7413-7417 (1987); P. Hawley-Nelson. WO 94/05624 et al., J. P. Behr, EP 0394111).
The individual reagents used for this are very varied. This is mostly due to the fact that they have been found empirically. Thus it has not been possible to specifically provide a compound for the application in question. Moreover the fact that compounds exist which only have an effect in a mixture with other reagents reveals the whole complexity of transfection. A review of most of the reagents that have been found up to now is given by Behr (Bioconjugate Chem., 5, 382-389 (1994) and the references cited therein). However, in general the majority of the reagents can form liposomes.
The mechanism of transfection by cationic amphiphiles is still little understood. It appears plausible that the liposomes by means of their positive charge form a more positively charged complex with the DNA and this attaches itself to the negatively polarized cell membrane (P. L. Felgner, Nature 337, 387-388 (1989)). However, it is not certain how the penetration of the cell membrane and transport to the cell nucleus takes place.
Nevertheless, apart from the transfection efficiency it is possible to define a series of other requirements for new reagents:
for efficiency it should not be necessary to pretreat the cells such as by permeabilizing the cell membrane with DMSO, a detergent such as digitonin or by scraping. PA1 the reagents should not be toxic if possible especially not at the most effective concentration and should be preferably biologically degradable PA1 it should be possible to use them equally for all cells that come into consideration PA1 they should not have a specificity for certain DNA molecules PA1 it should also be possible to use them in vivo. This means that in addition to toxicity, compatibility with serum is also of major significance. This has often been the reason for a drastic decrease in the transfection efficiency. PA1 A.sub.1 represents an O--R.sub.2 group in which R.sub.2 has the meaning stated for R.sub.1 and can be the same as or different from R.sub.1 PA1 A.sub.2 represents an NR.sub.3 X or an N.sup.+ R.sub.3 R.sub.4 R.sub.5 Y.sup.- residue in which PA1 Y is a pharmaceutically acceptable anion, PA1 B.sub.1 is an NH[C(O)--(CH.sub.2).sub.p --NH].sub.q --Z residue in which p is a number from 1 to 6 and q is a number from 0 to 2, PA1 B.sub.2 can have the meaning stated for A.sub.1 and the meaning for A.sub.1 is only valid with B.sub.1 and that of A.sub.2 is only valid with B.sub.2.
Hence the object of the invention was to provide appropriate reagents which equally fulfill these requirements.